Genome Wide Regulation

Question 1

GeneArrays

Answer 1

used to measure the amount of mRNA from all the genes in an organism

Question 2

RNA-seq

Answer 2

Uses next generation DNA sequencing to determine transcriptome

Question 3

2-D gels

Answer 3

used to measure the amount of proteins expressed

Question 4

Mass-spectroscopy

Answer 4

identifies proteins

Question 5

Gene Arrays

Answer 5

• Each gene of a sequenced organism is fixed to a slide in an ordered “array”
• the army is then probed with mRNA from your cell (two conditions)

Question 6

Gene Array Technique

Answer 6

• RNA Isolation
• cDNA Generation
• Labeling of Probe
• Hybridization to Array
• Imaging

Question 7

Campylobacter has how many genes

Answer 7

1600

Question 8

Making the array

Answer 8

• genes or portions of genes are spotted in duplicate on glass slides by robots.

Question 9

Probing the array

Answer 9

• microarrays are good at comparing transcription profiles between two different conditions
• First, you make cDNA copies of the RNA with reverse transcriptase
• cDNA from one condition is labeled red dye
• The other condition is labeled with green dye
• Both sets are then hybridized to the array, and a machine detects the color.

Question 10

Gene Arrays

Answer 10

• then probed the away
• each gene on there twice
• yellow-both genes expressed

Question 11

Heat Map

Answer 11

• shows how much red versus gene per green
• repeat experiment and swap dyes looking for exact opposite effect
• fold changes never really greater than 3-4
  
  • only so green.

Question 12

RNA seq

Answer 12

• total RNA
• eukaryote (enrich mRNA by oligo(dT))
• prokaryote (remove RNA by kit)
• RNA fragmentation
• Random hexameter primed cDNA synthesis
• Ends repairment, adapter ligation, and PCR amplification
• Hiseq 2000 91PE or 101PE sequencing

Question 13

Flagella discussion

Answer 13

• RpoN mutant
• RNA isolated from both conditions
  
  • DNA digested by DNase
  • rRNA depleted
• cDNA made with random primers
• RpoN required for Flagella (class II genes)
• cjc242 is a host invasion space gene that uses hook apparatus to invade host cells

Question 14

Proteomics

Answer 14

the study of all the proteins in a cell

Question 15

advantages of proteomics

Answer 15

detects non-transcriptional control, can be cheaper

Question 16

disadvantage of proteomics

Answer 16

cheap methods miss low abundance proteins

Question 17

1st dimension of 2D electrophoresis

Answer 17

• separates by charge (isoelectric focusing)
• the charge on a protein depends on PH
• isolelectric point is pH where they have no charge
• proteins with positive charge will migrate toward negative electrode until they lose enough protons to lose charge

Question 18

2nd dimension of 2D electrophoresis

Answer 18

• separates by size (SDS PAGE)

- each spot corresponds to size

Question 19

Ahpc

Answer 19

highly expressed protein

Question 20

NapA

Answer 20

compensates for loss of Ahpc

Question 21

determining the spots

Answer 21

• spots that are regulated are cut out of the gel and extracted
• to determine protein identity:
  
  • Do N-terminal sequencing
  • determine the mass of protein by mass spectrometry.

Question 22

Tryptic Digest

Answer 22

• trypsin cuts proteins after argentine and lysine residues
• excise the spot, treat the spot with trypsin to cut it up
• weight fragments by mass spectroscopy

Question 23

LC-Maldi TOF

Answer 23

• Tags differ by 1 amu
• mass to charge ration (M/Z) is determined by the amount of time it takes to get to the detector - Time of Flight
• keep the charge 1, you get the mass
• time to get from pusher to detector tells you how big
• fly for about 12 feet

Question 24

Mass spectrum

Answer 24

every fragment that comes through we weigh

Question 25

computer puts together the sizes

Answer 25

the computer program determines the sizes of all the proteolytic fragments from the predicted genome sequence

• computer compares experimental mass spectrum to the predicted mass spectrum from the database
• identifies the protein that the fragment came from.